The present invention relates to a purified plant cytokinin oxidizing enzyme (ckx1) from Zea mays, the complete amino acid sequence of which has been elucidated, and to isolated nucleotide sequences encoding the enzyme. The invention further relates to novel methods for moderating the concentration of the enzyme and similar enzymes in plants in order to affect plant cell growth and death. Applications of the invention include the regulation of the production of ckx1 in plant roots to affect pathogenesis, the regulation to alter plant habit, and the bulk production of ckx1 enzyme for use in a plant biochemical assay.
Plant cytokinins are a class of plant hormones which, when combined with auxin, control cell division, promote shoot development from callus, release lateral buds from dormancy, and regulate plant structure and growth in a variety of ways. The naturally occurring active cytokinins in most higher plants are free-base zeatin (6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine) (hereinafter Z), and its 9-riboside (hereinafter ZR). Plant tissues normally contain, therefore, Z, ZR, and smaller amounts of N6-(xcex942-isopentenyl)adenine (hereinafter, iP) derived from biosynthetic precursors. Elevated cytokinin levels are associated with the development of seeds in higher plants, and have been demonstrated to coincide with maximal mitotic activity in the endosperm of developing maize kernels and other cereal grains. Exogenous cytokinin application (via stem injection) has been shown to directly correlate with increased kernel yield in maize. In addition, plant cells transformed with the ipt gene from Agrobacterium tumefaciens (encoding a dimethylallylpyrophosphate:5xe2x80x2-AMP transferase capable of increasing cellular production of Z and ZR) showed increased growth corresponding to an increase in endogenous cytokinin levels upon induction of the enzyme. Thus, given the biosignificance of cytokinins to the growth of plants, the ability to manipulate cytokinin levels in higher plant cells is of great commercial and scientific interest.
The action of cytokinin oxidase is a major method of effective cytokinin catabolism in plant cells. This inactivation of cytokinin is accomplished by the oxidative removal of the side chain from cytokinin free bases (or their ribosides) in the presence of molecular oxygen. An example of this reaction with iP is shown in FIG. 1a. Although the exact chemical mechanism for this reaction is unknown, it is suspected that the enzyme is reduced during the deprotonation of iP to N6-(xcex942-isopentenylimino)purine. The purine is then hydrolyzed into adenine and intermediate 3-methyl-2-butenal (FIG. 1b).
While the electron acceptor responsible for reoxidizing the reduced enzyme in plant cells is not known, molecular oxygen can do so in vitro. Alternatively, the reduced enzyme may be reoxidized in vitro by intermediates such as Cu+2/imidazole complexes or the artificial electron acceptor dichlorophenolindophenol (DCPIP).
Cytokinin oxidases are known to remove cytokinins from plant cells after cell division, and have also been postulated to be involved in the senescence process. Cytokinin oxidase activities have been shown to positively correlate to the mitosis of endosperm cells in maize kernels, along with the increase in natural cytokinin concentrations. Oxidase activity increases shortly after the increase in endogenous cytokinin levels. A similar correlation was demonstrated with artificially increased cytokinin levels in transgenic tobacco. Thus, expression of cytokinin oxidases is thought to be involved in the maintenance of hormonal homeostasis in developing plant cells. Because cytokinin oxidases appear to be substrate-inducible, they act in a negative regulatory fashion to reduce elevated cytokinin levels back to basal values. This substrate induction of cytokinin oxidase activity is a significant barrier to potential commercial applications which attempt to manipulate cytokinin levels in transgenic plants through increased cytokinin production.
Cytokinin oxidases have been discussed for a number of plant species, including Vinca rosea, beans (Phaseolus vulgaris and lunatus), wheat (Triticum aestivum), tobacco (Nicotiana tabacum), Dianthus caryophyllus, soy (Glycine max), and maize (Zea mays). All of these plant cytokinin oxidases have a similar substrate preference for iP and Z, but show limited or no reactivity with bulky, reduced, or aromatic side chain cytokinins. All also exhibit enhanced activity in the presence of copper plus imidazole. However, these enzymes show substantial variation in both specific activity and molecular weight. This is thought to be linked to the occurrence of glycosylated and unglycosylated variants of the protein, both between and within species.
In the case of the glycosylated cytokinin oxidase, the heavily glycosylated protein may present a carbohydrate-rich surface, preventing antibody formation against peptide epitopes. The glyco-epitopes to which antibodies are raised under these conditions are non-specific, and may prevent isolation of the protein, or clones containing the gene which encodes it, via immuno-chromatography or other immunology-based means. An earlier reported attempt to isolate the gene for maize cytokinin oxidase (ckx1) by immunoscreening of maize cDNA library expression products (Burch, 1992) was unsuccessful.
As demonstrated, the full amino acid sequence and encoding DNA for a cytokinin oxidase has been a long sought after goal in modern plant physiology.
It is therefore an object of the present invention to provide a means by which recombinant cytokinin oxidase may be produced in quantity so that the effects of cytokinin oxidase on plant growth and metabolism may be studied. It is also an object of the present invention to provide a means for the modification of cytokinin oxidase production in plant cells, in vivo, in order to modulate the endogenous cytokinin level of plant cells to effect altered pathogen resistance and plant growth properties.
The present invention, therefore, is directed to a novel, isolated and substantially purified plant cytokinin oxidizing enzyme, (ckx1), having a molecular weight most preferably of about 60 kD, a sequence length of from about 505 to 565 amino acid residues, preferably 525 to 545 amino acid residues, and most preferably 534 amino acid residues, and having cytokinin inactivating activity. The present invention is also directed to a protein having an amino acid sequence which includes the amino acid sequence of ckx1 (SEQ. ID NO. 1). The invention is directed as well to a protein which has cytokinin inactivating activity and which includes a portion of the amino acid sequence of ckx1 at least about 20 amino acid residues in length, where the included portion of the ckx1 sequence confers the cytokinin inactivating activity on the protein. The invention is directed to proteins which have cytokinin inactivating activity and have at least about 65% sequence identity to ckx1 and most preferably at least about 95% sequence identity to ckx1, with the remaining amino acids being conservatively substituted.
The invention is directed, moreover, to substantially isolated nucleic acid polymers encoding ckx1 or a cytokinin oxidizing homolog thereof. The nucleic acid polymer most preferably has a nucleic acid sequence of SEQ. ID NO. 3 or the predictable variants thereof described in SEQ. ID NO. 10. The invention is also directed to a substantially isolated nucleic acid polymer which contains a portion of SEQ. ID NO. 2, SEQ. ID NO. 3, or a nucleic acid polymer described by SEQ. ID NO. 10, the portion being at least 60 bp in length. In addition, the invention is directed to nucleic acid polymers which are able to hybridize with SEQ. ID NO. 2, SEQ. ID NO. 3, or a nucleic acid polymer described by SEQ ID NO. 10, under conditions of 0.5xc3x97to 2xc3x97SSC buffer, 0.1% SDS, and a temperature of 55-65xc2x0 C. Nucleic acid polymers which encode cytokinin oxidases and meet the above requirements encode proteins of sufficient similarity to ckx1 to be generally recognized as equivalents of ckx1 among those skilled in the biochemical arts.
The invention is also directed to a host cell incorporating a vector containing the aforementioned DNA, and to a method for producing ckx1 or a homolog thereof using such a host cell. The method preferably comprises first ligating DNA encoding the aforementioned ckx1 or a segment or homolog thereof and an appropriate promoter (such as the PB7 root-specific promoter, Conkling, M. A., et al., U.S. Pat. No. 5,459,252; or CaMV35S promotor, Odell, 1985), or a combination of promoters (Hoffman, U.S. Pat. No. 5,106,739) 3) into an appropriate DNA vector (for instance, pBIN19 for use in Agrobacterium tumefaciens). The vector construct may then be directly transformed into a host cell, such as Pichia pastoris (described in Example 2). It may also be incorporated into a secondary vector for transformation into a host cell, such as Agrobacterium tumefaciens, and transformed into a plant cell host (described in Example 4 with Nicotiana tabacum).
Alternatively, for production of larger amounts of the enzyme, the DNA encoding ckx1, or a portion thereof, may be transformed into Pichia, according to the methods described in Example 2, or in Su, et al., 1996 and Skory, et al., 1996. When transformed into Pichia spp., ckx1 is secreted into the culture medium because of the presence of a secretory signal peptide at the N-terminus of the ckx1 coding region. Thus, active enzyme may be readily purified from bulk Pichia cultures without a lysing step. ckx1 produced in such a manner may be used in a biochemical assay to determine unknown concentrations of cytokinin in biological samples, according to the method of Example 3.
A plant host cell generated by the above method may be regenerated into an entire plant. Depending on the promoter used in the vector construct, ckx1 may be produced constitutively or by induction through natural or artificial environment factors. Plants transformed with vectors containing tissue-specific or trauma-specific promoters and a sequence encoding ckx1 can exhibit altered resistance to certain cytokinin-linked plant pathologies, such as infection by certain nematodal or fungal species.
The discoveries described herein provide an important analytical tool for, and a critical link in, the development of methods by which the manipulation of cytokinin oxidase activity may be used to either inhibit or enhance a variety of cell growth functions in plants in a desired manner. Possible uses include the development of commercial plants with increased grain production, disease resistance, or with more desirable secondary growth characteristics. The enzyme and its encoding nucleic acids have important uses in the study of plant cell growth cycles and senescence. In addition, many other pharmaceutical and agricultural uses for ckx1 and its gene may be discovered. The enzyme, methods of expressing the enzyme, and methods for its use are described in greater detail below.
Other features and objects of the present invention will be in part apparent to those skilled in the art and in part pointed out hereinafter.